Università degli Studi di Cagliari
Dipartimento di Scienze della Vita e dell’Ambiente Sezione di Scienze del Farmaco
DOTTORATO DI RICERCA
Scienze e Tecnologie Farmaceutiche Ciclo XXVI
Secondary Metabolites from Otanthus maritimus, Stachys glutinosa and Withania somnifera: Isolation, Structure Elucidation and
Interactions with Cannabinoid and Opioid Systems Settore scientifico disciplinare di afferenza
CHIM/08
Presentata da: Nicola Anzani Coordinatore Dottorato Prof. Elias Maccioni
Tutor Dott. Filippo Cottiglia anno accademico 2012 – 2013
II
ACKNOWLEDGEMENTS
I am most grateful to my supervisor, Dr. Filippo Cottiglia, for general help and many fruitful discussion about my work. To him goes all my gratitude for being always an example at work and for his continuous support and encouragement.
Special thanks to Dr. Stefania Ruiu and Dr. Alessandro Orrù (Institute of Translational Pharmacology, UOS of Cagliari, National Research Council, Parco Scientifico e Tecnologico, Pula, Italy) for binding assays and analgesia experiments.
Thanks are also due to Dr. Simona Distinto (Department of Life and Environmental Sciences, Cagliari) for molecular modeling studies. I wish to thank Dr. Marco Leonti (Department of Life and Envi-‐ ronmental Sciences, Cagliari) for the identification of the plant ma-‐ terial.
I am grateful to Dr. Amit Agarwal (Natural Remedies Pvt. Ltd., Bangalore, India) for providing the methanol extract of Withania
somnifera roots.
I would like to express my gratitude to my colleagues at the La-‐
boratory of Medicinal Chemistry of the Department of Life and Enviromental Sciences for creating a friendly and stimulating working
atmosphere.
Last but not least, I would like to give special thanks to my family that always believed in me and to my girlfriend Michela for her infinite patience and love that allowed me to overcome all obstacles. You’re my lighthouse in the storm.
III
Nicola Anzani gratefully acknowledges Sardinia Regional Government for the financial support of her PhD scholarship (P.O.R. Sardegna F.S.E. Operational Programme of the Autonomous Region of Sardinia, European Social Fund 2007-‐2013 -‐ Axis IV Human Resources, Objec-‐ tive l.3, Line of Activity l.3.1.)”.
IV
TABLE OF CONTENTS
1. INTRODUCTION
12. OPIOID RECEPTORS
4 • 2.1 Natural Opioids Ligands 83. CANNABINOID RECEPTORS
14 • 3.1 CB1 Receptor 17 • 3.2 CB2 Receptor 19 • 3.3 Natural Cannabinoids ligands 204. AIM OF THE WORK
265. METHODOLOGY OF ISOLATION PROCEDURE
28• 5.1 Extraction 28 • 5.2 Fractionation 28
• 5.3 Vacuum Liquid Chromatography (VLC) 29 • 5.4 Purification 29
• 5.5 Open Column Chromatography 30
• 5.6 High Performance Liquid Chromatography 31
V
6. METHODOLOGY OF STRUCTURE ELUCIDATION
32• 6.1 Nuclear Magnetic Resonance Spectroscopy 32 • 6.2 Mass Spectrometry 35
7. BIOLOGICAL EXPERIMENTS
37• 7.1 Binding Assay 37 • 7.2 Tail Flick Test 38 • 7.3 Hot Plate Test 38
8. Othantus maritimus
40• 8.1 Botanical Decription 40
• 8.2 Geographical distribution and habitat 42 • 8.3 Use in Folk Medicine 43
• 8.4 Chemical Composition 43
• 8.5 Biological Actvity of N-‐alkylamides 48
9. Stachys glutinosa
51• 9.1 Botanical Decription 51
• 9.2 Geographical Distribution and Habitat 53 • 9.3 Use in Folk Medicine 53
• 9.4 Chemical Composition 54
• 9.5 The Phytochemical Investigation on Genus Stachys 55 o 9.5.1 Diterpenes 55
VI • 10.1 Botanical Description 62
• 10.2 Geographical Distribution and Habitat 62 • 10.3 Use in Folk Medicine 64
• 10.4 Pharmacological studies of W. somnifera extracts 65 • 10.5 Chemical Composition 66
o 10.5.1 Withanolides 66 o 10.5.2 Alkaloids 70
o 10.5.3 Biological Activities of Withania somnifera Withanolides 72
11. RESULTS
74• 11.1 Extraction of O.maritimus Roots 75
• 11.2 Isolation of Metabolites from O.maritimus 75
• 11.3 Structure Elucidation of Metabolites from O. maritimus 78
o 11.3.1 Structure Elucidation of Compound 1 78 o 11.3.2 Structure Elucidation of Compound 5 88 o 11.3.3 Strucure Elucidation of Compound 12 96 o 11.3.4 In silico Modelling Study 105
o 11.3.5 Structure Elucidation of Known Compounds 111 • 11.4 Extraction of S. glutinosa Aerial Parts 141
• 11.5 Isolation of Metabolites from S. glutinosa L. 141
VII
glutinosa 144
o 11.7.1 Structure Elucidation of Compound 22 144 o 11.7.2 Structure Elucidation of Known Compounds 159 • 11.8 Isolation of Metabolites from W. somnifera 174 • 11.8.1 Structure Elucidation of Known Compounds 177
12. BIOLOGICAL RESULTS
195• 12.1 Opioid and Cannabinoid Binding Affinity of Compounds Isolated from O.maritimus 195
• 12.2 Opioid Binding Affinity of Compounds Isolated from
S.glutinosa 200
o 12.2.1 Effects of xanthomicrol on morphine-‐induced Analgesia 202
o 12.2.2 Receptor Binding Affinity of Methanol and Alkaloid Extract from W.somnifera (WSE and WSAE) 205
o 12.2.3 Analgesia Experiments 206
o 12.2.4 Effects of WSE on Morphine-‐induced Analgesia 206 o 12.2.5 Effect of WSE on Morphine-‐induced Hyperalgesia 212 o 12.2.6 Effect of WSME on Morphine-‐induced Hyper-‐locomotion
214
VIII
14. EXPERIMENTA
LSECTION
221• 14.1 General Experimental Procedures 221 • 14.2 O. maritimus Plant Material 222
• 14.3 O. maritimus Extraction and Isolation 222
• 14.4 O.maritimus Analytical and Spectroscopic Data of the New Compounds 225
• 14.5 Stachys glutinosa Plant Material 226
• 14.6 Stachys glutinosa Extraction and Isolation 226 • 14.7 Semi-‐synthesis of 5-‐demethyltangeretin (23) 228 • 14.8 Semi-‐synthesis of Tangeretin (24) 228
• 14.9 W. somnifera Plant Material 229
• 14.10 W. somnifera Extraction and Isolation 229
o 14.10.1 Extraction and Separation Procedure of Alkaloids 229 o 14.10.2 Separation Procedure of Withanolides 231
15. MOLECULAR MODELING
233• 15.1 Ligands Preparation 233 • 15.2 Protein 233
• 15.3 Docking and Post-‐Docking Experiments 234
16. BIOLOGY ASSAY
235• 16.1 Animals 235
IX • 16.3 [ H]-‐DAMGO-‐[ H]-‐DPDPE (opioid receptors) Binding Assay
237
• 16.4 [3H]-‐CP-‐55,940 (cannabinoid receptors) Binding Assay 238 • 16.5 [3H]-‐Muscimol (GABAA receptor) Binding Assay 240
• 16.6 Analysis of Samples 241
17. ANALGESIA EXPERIMENTS
243• 17.1 WSE Tail-‐flick and Hot-‐plate Test 243 • 17.2 Xantomichrol: tail-‐flick test 244
• 17.3 Morphine-‐induced Hyperalgesia Experiment 245 • 17.4 Spontaneous and Morphine-‐induced Motor Activity
Experiments 245 • 17.5 Data analysis 246
18.
REFERENCES
24819.
PUBLICATIONS AND PRESENTATIONS
265X
ABBREVIATIONS
ACN acetonitrile
APT attached proton test
CB cannabinoid
CDCl3 deuterated chloroform
CD4O deuterated methanol
CPP conditioned place preference
d doublet
DCM dichloromethane
DEPT distortionless enhancement by polarization tranfer DQF-‐COSY double-‐quantum filtered correlation spectroscopy DOR δ opioid receptor
ESI MS electrospray mass spectrometry EtOAc ethyl acetate
FDA food and drugs administration GABA gamma-‐aminobutyric acid
[3H]-‐DAMGO [(D-‐Ala2, N-‐Me-‐Phe4, Gly5-‐ol-‐) enkephalin] [3H]-‐DPDPE [(D-‐Pen 2,5)-‐enkephalin]
HMBC heteronuclear multiple bond correlation HPLC high performance liquid chromatography
XI HSQC heteronuclear single quantum coherence
IC50 inhibition concentration (50% inhibition)
µg microgram
µl microliter
Ki inhibition constant
KOR k opioid rceptor MeOH methanol
MOR µ opioid receptor NMDA N-‐methyl-‐d-‐aspartate NMR nuclear magnetic resonance NP normal phase
OME Otanthus maritimus extract
PAg Periaqueductal grey PMFs polymethoxyflavones
RP reversed phase s singlet
spp species TOF time of flight
THC tetrahydrocannabinol
t triplet
XII TLC thin layer chromatography
SGE Stachys glutinosa extract
UV ultraviolet
VLC vacuum liquid chromatography WSAE Withania somnifera alkaloid extract
WSE Withania somnifera methanol extract
1
1. INTRODUCTION
Plants have been used for thousands of years to treat diseases and today too, they are the almost exclusive source of drugs for the majority of the world’s population.
In the 19th century, with the isolation of morphine from opium, it was
begun to employ the pure active ingredients rather than whole extracts.1 After the discovery of morphine a lot of plant-‐originated
drugs have been discovered and various secondary metabolites are currently in use such as, for example, quinine from Cinchona species, cardiac glicosides from Digitalis purpurea, vinblasine and vincristine from Catharanthus roseus, taxol from Taxus brevifolia and the antimalarian compound, artemisinin, from Artemisia annua.
Higher plants are also an important source of drugs that act as agonist to opioid receptors and among all, morphine, isolated from the opium poppy, Papaver somniferum. Morphine is a µ opioid receptor agonist and is the most potent analgesic currently used in clinic for the treatment of moderate or severe pain. Salvinorin is another receptor opioid ligand (k agonist) isolated from plants (Salvia divinorum) but it does not show any analgesic activity and has been classified as a hallucinogenic agent.2
2 reported to contain secondary metabolites that interact with the endocannabinoid system. These compounds, also called phytocannabinoids, are capable of either directly interacting with cannabinoid receptors (CB1 and CB2) or sharing chemical similarity
with cannabinoids or both.3 Δ9-‐tetrahydrocannabinol (Δ9-‐THC) from
Cannabis sativa is a non-‐selective agonist to cannabinoid receptors
and is used for the treatment of neuropathic pain or for refractory forms of treatment with morphine derivatives.4
Recently, N-‐alkylamides from Echinacea spp. have been identified as
CB2 receptor selective agonists and are responsible of the
immunomodulatory effect of this plant.5
Powerful new technologies such as high-‐throughput screening and combinatorial chemistry dramatically increase the possibility of drug discovery. Nevertheless, natural products still offer unmatched structural variety when compared to synthetic compounds. For example, natural products are more likely to be rich in stereochemistry and concatenated rings than the structures obtained by the combinatorial libraries.
To date, natural products still represent a very important source in the discovery and development of new medicines and a significant part of the therapeutic armamentarium of doctors is represented by
3 natural medicines or natural-‐derived products.6 If we consider the
new drugs approved in 2010 by Food and Drugs Administration (FDA), half of the 20 fully approved small molecules were natural products or directly derived therefrom, confirming the importance of natural products as source of new drugs.6
4
2. OPIOID RECEPTORS
The term opioid applies to any substance that produces effects similar to those of morphine and that are blocked from specific antagonists (naloxone). Among these there are natural alkaloids, synthesis or semisynthesis compounds, endogenous opioid peptides. These substances act on specific receptors of the peripheral and central nervous system (that take the generic name of opioid receptors) acting mainly as modulators of the painful sensations but also through specific transcription factors nuclear receptors. The term opioid is frequently used improperly to indicate, more restrictively, the alkaloids that can be found in opium, a mixture of substances derived from the latex of Papaver somniferum, and their semi-‐ synthetic derivatives; the correct term to describe these substances is, instead, opiates. The evidence of the use of opium as a medicine as well as a substance for luxury, dates back to many centuries before Christ, given in Latin texts and Homer, and to Roman imperial and Republican.7 Opioids act on a family of receptors in the central and peripheral nervous system, which includes four subtypes: μ opioid receptor (MOR), δ opioid receptor (DOR) and κ opioid receptor (KOR). All these receptors belong to the superfamily of G protein-‐coupled
5 receptors. The activation of these receptors leads to: inhibition of adenylate cyclase and thereby reduced synthesis of cAMP, the inhibition of Ca2+ channels that results in a reduction in the release of
neurotransmitter, the opening of K+ channels that results in
hyperpolarization of the membrane and reduction of nerve activity.7 G proteins involved in signal transduction are Gi (inhibitor). These cellular effects are reflected in a wide variety of physical symptoms such as analgesia and sedation, sleep induction, respiratory depression (caused by opioid action at the level of the bulbar respiratory centre sensitive to arterial pCO2), central nervous depression, gastrointestinal motility inhibition and inhibition of the cough reflex.7 All opioid receptors modulate the analgesic action although they operate at different levels. MOR: generating analgesia (sovraspinal level), miosis, and respiratory depression, decrease in gastrointestinal activity, euphoria; KOR: produces analgesia (spinal level), miosis, and respiratory depression, dysphoria (unlike μ receptors); DOR: no analgesia, but decreases the intestinal transit and depresses the immune system.7 Opioids tend to inhibit neuronal transmission at both pre and post synaptic level. In fact, the activation of presinaptic µ receptors causes inhibition of N-‐type calcium
channels and thus a reduction in the production of neurotransmitters, while the activation of µ postsynaptic receptors produces
6 calcium L-‐type.
MOR is the most widespread receptor and mediate most of the pharmacological effects of opioid analgesics.
Physiologically active molecules on these receptors are the endogenous opioids peptides, β-‐ endorphins, dynorphines A and B, and enkephalins, endogenous substances better defined as opioid peptides which are synthesized respectively starting from large precursor peptide, proopiomelanocortin, proenkephaline and prodinorphine, splitting by specific endopeptidase.7
The endogenous opioids, β-‐endorphins, dynorphines A and B, and enkephalins exert their analgesic action at spinal and sovraspinal level. They also cause analgesia with a peripheral action mechanism associated with the inflammatory process. In the central nervous system, opioids exert an inhibitory action on neurotransmitters. At sovraspinal level, activation of opioid receptors inhibit neuronal activity and therefore the release of noradrenaline from the locus
coeruleus and nucleus reticularis paragigantocellularis (NRPG), and
the release of serotonin from nucleus Raphe Magnus (NRM), with inhibition of pain transmission. MOR agonists prevent the release of the inhibitory transmitter GABA activating the Periaqueductal grey (PAG) systems that regulate the activity of the bulb.8 In particular, the
7 GABA transmission can have opposite effects on pain processing in relation to its location within the central nervous system; its activation causes analgesia at spinal level, while it is pronociceptive at sopraspinal level.9,10 The systemic administration of GABA
A and GABAB
agonists such as benzodiazepines increases the opioid-‐induced analgesia11,12 and attenuates the development of tolerance.13
Opioids also exert a neuromodulator action of pain signal on afferent neurons located in the dorsal horn of the spinal cord and neuronal interconnection paths for pain signal transmission in the brain.
At spinal level, the activation of k and µ receptors blocks the release of substance P, peptide released following a skin lesion from the fibers relating to the rear horns of the spinal cord. Substance P is a neurotransmitter of the anguished transmission, so blocking its release also locks the transmission of pain information.14
Glutamate is the primary excitatory neurotransmitter involved in the transmission of nociceptive stimuli at spinal level.10 In addition, N-‐ Methyl-‐D-‐Aspartate (NMDA) receptor sensitization on spinal neurons play a key role in the development of tolerance induced by opioids.8 Consistently, the co-‐administration of NMDA receptor antagonists enhances the opioid-‐induced analgesia.15
8
2.1 Natural Opioids Ligands
As mentioned before, the investigation of natural products has proven to be an excellent source of clinical agents for a number of therapeutic areas including pain.6
Morphine (Figure 1) is the most abundant opiate found in opium (8-‐ 14% of dry weight), the dried latex is obtained by shallowly slicing the unripe seedpods of the Papaver somniferum poppy. Morphine was the first active principle purified from a plant source and is one of at least 50 alkaloids of several different types present in opium.
9
Morphine is primarily used to treat both acute and chronic severe pain for example in myocardial infarction, in cancer pain and for labour pains.7
In fact like other opioids, it acts directly on µ receptor of the central nervous system (CNS) to relieve pain. Morphine has a high potential for addiction; tolerance and psychological dependence develop rapidly. Tolerance to respiratory depression and euphoria develops more rapidly than tolerance to analgesia, and many chronic pain patients are being maintained on a stable dose, for many years.7
In addition morphine acts on the myenteric plexus in the intestinal tract, reducing gut motility, causing constipation. The gastrointestinal effects of morphine are mediated primarily by µ receptors in the bowel.7
New natural therapies are currently being explored as analgesic potential alternatives to morphine and derivatives.17
Kratom (Mitragyna speciosa Korth., Rubiaceae) is an indigenous herb of Southeast Asia that is traditionally used to treat fever, diarrhea, fatigue, pain, and as a substitute for morphine in treating opioid addicts. The main component of kratom is the indole alkaloid mitragynine (Figure 2), which has been reported to have affinities for all three opioid receptors, though it appears to be relatively selective
10 derivatives of mitragynine, as pseudoindoxyl and 7-‐ hydroxymitragynine, have also been found to have affinity for opioid receptors (Figure 2).17
mitragynine pseudoindoxyl-‐mitragynine 7-‐hydroxymitragynine
Figure 2. Structures of Mitragyna speciosa alkaloids
Research interest in mitragynine stems from its increasing use as a remedy for opioid withdrawal by individuals who self-‐treat chronic pain. In addition this compound is known to produce antinociception in mice in the hot-‐plate and in the tail-‐flick tests.17 However, the exact mechanisms underlying the effect of mitragynine are currently unknown. It has been hypothesized that the MOP agonism of mitragynine might avert withdrawal symptoms, while KOP agonism
11 might attenuate reinforcement and blunt cravings. The collective findings of the effects of mitragynine indicate that the molecule and its derivatives may be useful for the development of new analgesics and possibly for the treatment of opioid abuse.17
Selective KOP agonists are also capable of producing clinically useful analgesia, but lack the respiratory depression, constipation, and addictive properties associated with MOP agonists. However, a side effect associated with activation of KOP receptors is dysphoria. Still, KOP agonists are targets for achieving pain relief without the negative side effects associated with MOP agonists. Although KOP agonists are known to produce dysphoric effects, there is still some hope that a clinically useful analgesic may be found.17
Salvinorin A, a neo-‐clerodane diterpene (Figure 3), is the active hallucinogenic component in the Mexican mint plant Salvia divinorum (Lamiaceae). This plant has been used by the Mazatec Indians in Oaxaca, Mexico, as a hallucinogenic agent, and to relieve diarrhea, headache, and rheumatism.
12
Figure 3. Structure of salvinorin A
However, until recently, the target of the hallucinogenic effects was not clear, as Salvinorin A lacks activity at the targets of other known hallucinogens, specifically serotonin receptors, cholinergic receptors, and cannabinoid receptors.
In 2002, Salvinorin A was identified as a potent and selective KOP agonist. This result is surprising considering that Salvinorin A lacks the basic nitrogen that has long been thought to be required for opioid activity. However, given the known hallucinogenic effects of other KOP agonists, this finding is not unprecedented. Salvinorin A produces a discriminative effect in both rats and non-‐human primates that is similar to other KOP agonists. It has also been shown to produce analgesia in mice that can be blocked by a KOP receptor antagonist.17
13 Another natural opioid is ibogaine (Figure 4), an indole alkaloid isolated from the root, root-‐bark, stems, and leaves of the African shrub Tabernanthe iboga. This plant has been used by indigenous people in low doses to combat fatigue and hunger and in higher doses as a sacrament in religious rituals. The psychopharmacology of ibogaine is complex due to its affinity for several receptors, transporters, and ion channels. In addition, its primary metabolite, 12-‐ hydroxyibogamine, is also biologically active. The most-‐studied therapeutic effect of ibogaine is the reduction or elimination of addiction to opioids. The mechanism by which ibogaine exerts its anti-‐ addictive effects is presently unknown although several receptor systems have been implicated in its activity. However, it has been speculated that its k agonist actions contribute to its effects on stimulant self-‐administration and analogs of ibogaine are currently being explored as potentially safer medications.18
14
3. CANNABINOID RECEPTORS
The cannabinoid receptors are a class of cell membrane receptors under the G protein-‐coupled receptor superfamily19-‐21 which contain
seven transmembrane spanning domains.22
There are currently two known subtypes, termed CB1 and CB223,24. (Figure 5). The CB1 receptor is expressed mainly in the brain, but also in the lungs, liver and kidneys. The CB2 receptor is expressed mainly in the immune system and in hematopoietic cells.25
15 Cannabinoid receptors are activated by three major groups of ligands, endocannabinoids (such as anandamide and 2-‐arachidonoylglycerol (2-‐AG) (Figure 6), phytocannabinoids (such as Δ9-‐THC and alkylamides,
found in Cannabis and Echinacea species, respectively) (Figure 7) and synthetic cannabinoids (such as HU-‐210). All of the endocannabinoids and phytocannabinoids are lipophilic, i.e. fat soluble, compounds. Cannabinoids bind reversibly and stereo-‐selectively to the cannabinoid receptors. (1) (2)
Figure 6. Structures of anandamide (1) and 2-‐arachidonoylglycerol (2)
16
Figure. 7 Structure of Δ9-‐THC
After the receptor is engaged, multiple intracellular signal transduction pathways are activated. At first, it was thought that cannabinoid receptors mainly inhibited the enzyme adenylate cyclase (and thereby the production of the second messenger molecule cyclic AMP), and positively influenced inwardly rectifying potassium channels (=Kir or IRK). However, a much more complex picture has appeared in different cell types, implicating other potassium ion channels, Ca2+ channels, protein kinase A and C, Raf-‐1, ERK, p38, c-‐ fos, c-‐jun and many more.26
Separation between the therapeutically undesirable psychotropic effects, and the clinically desirable ones has not been reported with agonists that bind to cannabinoid receptors. Δ9-‐THC, as well as the
two major endogenous compounds identified so far, anandamide and 2-‐arachidonylglycerol, that bind to the cannabinoid receptor, produce
17 most of their effects by binding to both the CB1 and CB2 receptors. While the effects mediated by CB1, mostly in the central nervous system, have been thoroughly investigated, those mediated by CB2 are not equally well defined.27
3.1 CB1 Receptor
CB1 receptors are expressed most densely in the central nervous system and are largely responsible for mediating the effects of cannabinoid binding in the brain.
The analgesic effects of cannabinoids are based on the interaction of these compounds with CB1 receptors on spinal cord interneurons in the superficial levels of the dorsal horn. Signals on this track are also transmitted to the periaqueductal gray (PAG) of the midbrain. Endogenous cannabinoids are believed to exhibit an analgesic effect on these receptors by limiting both GABA and glutamate of PAG cells that relate to nociceptive input.28
They are also found in other parts of the body. For instance, in the liver, activation of the CB1 receptor is known to increase de novo lipogenesis.29 Activation of presynaptic CB1 receptors is also known to
18 shock.30
Inhibition of gastrointestinal activity has been observed after administration of Δ9-‐THC or anandamide. This effect is assumed to be
CB1-‐mediated, since this receptor is expressed by the peptide hormone cholecystokinin, and application of the CB1-‐specific antagonist SR 141716A Rimonabant blocks the effect.31
Cannabinoids are well known for their cardiovascular activity. Activation of peripheral CB1 receptors contributes to hemorrhagic and endotoxin-‐induced hypotension. Anandamide and 2-‐AG, produced by macrophages and platelets, respectively, may mediate this effect.32
Many studies suggest that the effects of endocannabinoids on memory are dependent on what type of neurons are being targeted (excitatory vs. inhibitory) and the location of these networks in the brain.33
Evidence for the role of the endocannabinoid system in food-‐seeking behavior comes from a variety of cannabinoid studies. Emerging data suggest that Δ9-‐THC acts via CB1 receptors in the hypothalamic nuclei to directly increase appetite.34
19
3.2 CB2 Receptor
CB2 receptors are found throughout tissues of the spleen, tonsils, and thymus gland mainly expressed on T cells of the immune system, on macrophages and B cells, and in hematopoietic cells. When activated, they too can affect the release of chemical messengers, in this case the secretion of cytokines by immune cells, and can in addition modulate immune cell trafficking.35 They are also expressed on
peripheral nerve terminals, playing a role in antinociception, or the relief of pain. In the brain, they are mainly expressed by microglial cells, where their role remains unclear.
To be specific, this receptor has been implicated in a variety of modulatory functions, including immune suppression, induction of apoptosis and of cell migration.36
Therefore, they are also expressed in the brain, though not as densely as the CB1 receptor and are located on different cells.37 Unlike the
CB1 receptor, in the brain, CB2 receptors are found primarily on microglia, but not on neurons.
CB2 receptors are also found throughout the gastrointestinal system, where they modulate intestinal inflammatory response. Thus, CB2
20 bowel diseases, such as Crohn's disease and ulcerative colitis.38,39
The endocannabinoid system, through CB2 signaling, plays a key role in the maintenance of bone mass: CB2 are expressed in osteoblast, osteocytes and osteoclast. CB2 agonists enhance endocortical osteoblast number and activity while restraining trabecular osteoclastogenesis. Another important effect is that CB2 agonists attenuate ovariectomy-‐induced bone loss while increasing cortical thickness. These findings suggest CB2 offers a potential molecular target for the diagnosis and treatment of osteoporosis.40
3.3 Natural Cannabinoids Ligands
Cannabis sativa have been used for centuries and are known to
produce an analgesic effect in addition to hallucinogenic effects such as feelings of dissociation from reality. Cannabinoids are divided into two categories: classical cannabinoids and non-‐classical cannabinoids.41
Classical cannabinoids are tricyclic dibenzopyran derivatives that are both natural and obtained by semisynthesis starting from the first one. This group of molecules is exemplified by Δ9-‐THC; the main
21 psychotropic principle of cannabis. Non-‐classical cannabinoids emerged from Pfizer SAR studies of the classical cannabinoids.41
These compounds are devoided of the dihydropyran ring present in Δ9-‐THC, as for example CP47497 (Figure 8).42
Δ9-‐THC acts as an agonist with efficacy similar to that of anandamide. It has been suggested that the hallucinogenic effects of Δ9-‐THC arise
from the compound’s ability to mimic the action of anandamide at cannabinoid receptors, while simultaneously antagonizing 2-‐AG at these same receptors. This hypothesis is supported by the observation that a single high dose of a CB1 receptor antagonist has only a limited ability to block the subjective effects of cannabis ingestion.43
Dronabinol44,48 is the pure isomer of Δ9-‐THC, which is the main isomer found in cannabis. It is sold as Marinol (Figure 8) and considered to be non-‐narcotic with low risk of physical or mental dependence. Marinol has been approved by the U.S. Food and Drugs Administration (FDA) for the treatment of anorexia in AIDS patients, as well as for refractory nausea and vomiting of patients undergoing chemiotherapy.
An analog of Dronabinol, Nabilon44,48 (Figure 8), is available
22 FDA approval and began being marketed in the U.S. in 2006.
Female cannabis plants contain more than 60 cannabinoids including cannabindiol, thought to be the major anticonvulsivant that helps multiplesclerosis patients45, and cannabichromene (Figure 8), an anti-‐
inflammatory which may contribute to the pain-‐killing effect of cannabis.46 It takes over one hour for Marinol to reach full systemic
effect47compared to seconds or minutes for smoked or vaporized
cannabis.48
Recent advances in the understanding of the endocannabinoid system have broadened the therapeutic possibilities resulting from its manipulation. CB1 receptor antagonists have received the most of the attention of the potential drugs affecting the endocannabinoid system. Their primary indication is for obesity. The rationale behind this indication lies in the generally accepted notion that ingestion of cannabis enhances the appetite, resulting in increased consumption of rich foods. Therefore, CB1 receptor antagonists should function to reduce the appetite, thereby reducing caloric intake and body weight.49,50 The first reported CB1 receptor antagonist was rimonabant (SR141716, Accomplia)51 (Figure 8) wich show nanomolar
23 Another indication for CB1 receptor antagonists is in the treatment of drug abuse. Several studies in animals have observed that CB1 receptor antagonists such as rimonabant reduce the rewarding properties of opioid receptor agonists.52-‐55 In fact, these rewarding
properties are absent in CB1 receptor knock-‐out mice.57 However,
opioid receptors do not seem to be involved in the hallucinogenic effects of CB1 receptor agonists, as opioid receptor antagonists do not block these effects.58 Also, the CB1 receptor seems to be involved
in responses to both nicotine and alcohol; CB1 receptor antagonists are able to block nicotine-‐induced conditioned place preference (CPP) and to decrease alcohol consumption.58 In October 2008, the European Medicines Agency’s Committee for Medicinal Products for Human Use (CHMP) had determined that the risks of Accomplia outweighed its benefits, and subsequently recommended the product be suspended from the UK market and doctors not prescribe the drug due to the risk of serious psychiatric problems and even suicide. Extracts of Cannabis are known to produce analgesic effect. In April 2005, Canadian authorities approved the marketing of Sativex, a mouth spray for multiple sclerosis patients, who can use it to alleviate neuropathic pain and spasticity. Sativex contains tetrahydrocannabinol together with cannabindiol and is a preparation of whole cannabis rather than individual cannabinoids.45 It is
24 based prescription drug in the world (in modern times). In addition, Sativex received European regulatory approval in 2010.
A particularly attractive feature of selective CB2 receptor agonists as therapeutics is that they are devoid of any known hallucinogenic effects such as those associated with CB1 receptor agonists.50
Since CB2 receptors are believed to play an important role in distinct pathophysiological processes, including metabolic dysregulation, inflammation, pain, and bone loss, they have, therefore, become of interest as new targets in drug discovery. Recently, some phytocannabinoids have been identified as selective CB2 agonist and, among all, a few fatty acid amides isolated from Echinacea purpurea that justify the use of Echinacea as herbal immunomodulators worldwide plant.59
Another phytocannabinoid that act as potent and selective CB2 agonist is the sesquiterpene β-‐caryophyllene.5 This compound have been identified in many food plants and also in Cannabis sativa L. essential oil. β-‐caryophyllene showed high oral bioavailabilty and strong anti-‐inflammatory and analgesic effects and may be considered a good candidate for clinical trials targeting the CB2 receptor.60
25
Dronabinol Nabilon Cannabindiol
CP47497 Rimonabant β-‐caryophyllene
Figure 8. Natural and synthetic cannabinoids structures
26
4. AIM OF THE WORK
In our continuous search for plant secondary metabolites that bind to CB and/or opioid receptors, we selected four extracts that showed interesting affinity versus the above mentioned receptors (Table 1). In particular: the DCM extract obtained from the aerial parts of Stachys
glutinosa (SGE) was able to bind with a good affinity both MOR and
DOR with a Ki of 10.3 and 9 µg/mL, respectively while the DCM extract
from the leaves of Otanthus maritimus (OME) showed good binding affinity to CB1 (Ki = 2.2 µg/mL) and CB2 (Ki = 1.3 µg/mL) and moderate
affinity to MOR and DOR. The third was an alkaloid fraction obtained from the MeOH extract of the roots of Withania somnifera (WSAE) that displayed appreciable affinity versus DOR (Ki = 25.5 µg/mL), CB1
(Ki = 23.5 µg/mL), CB2 (Ki = 20.3 µg/mL) and GABAA (Ki = 14 µg/mL).
The in toto MeOH extract (WSE) bound with very low affinity to CB and opioid receptors but displayed interesting affinity to GABAA
receptors (Ki = 14 µg/mL) (Table 1).
Based on this results this study, carried out in collaboration with the group of Dr. Stefania Ruiu of CNR-‐Institute of Translational Pharmacology of Cagliari, aimed to:
27 1. Isolate the secondary metabolites that were responsible of the
observed binding affinity
2. Identify the compounds by spectrometric and spectroscopic methods
3. Evaluate the binding affinity of the isolated metabolites to opioid and cannabinoid receptors.
4. Evaluate the most potent and abundant compounds in antinociceptive experiments in mice.
Table 1. Ki values of OME, SGE, WSE, and WSAE extracts for opioid,
cannabinoid, GABAA receptors
Receptor affinity (µg/ml) Extract µ δ k CB1 CB2 GABAA OME 10 ± 0.7 8.5 ± 1.3 - 2.2 ± 0.9 1.3 ± 0.3 - SGE 10.3± 0.2 9.0 ± 1 - - - - WSE 385 ± 14 166 ± 11 775 ± 56 837 ± 74 >1000 13 ± 2 WSAE 60 ± 7 25.5 ± 6 700 ± 120 23.5 ± 1 20.3 ± 2 14 ± 0.5
28
5. METHODOLOGY OF ISOLATION PROCEDURE
The isolation of a natural product can be divided into three main stages: extraction, fractionation, and purification.
5.1 Extraction
The first stage of the isolation procedure is the release of compounds from the cell mass and the removal of bulk of the biomass. Most of the bulk of biomass exists as fairly inert, insoluble, and often polymeric material, such as the cellulose of plants. The first step of the extraction is to release and solubilize the smaller secondary metabolites by a thorough extraction with an organic solvent or water. This can be done by a series of of stepwise extractions, using solvents of varying polarity, which acts as the first fractionation step, or by using a single solvent such as methanol, which should dissolve most natural products.
29 The second stage, the fractionation, consists to remove the most part of the unwanted material and to obtain a crude separation of the compounds mixture. Such step may involve vacuum liquid chromatography and liquid-‐liquid extractions.
5.3 Vacuum Liquid Chromatography (VLC)
VLC is a very convenient and simple chromatograpy method that is able to produce good resolution in short time. This technique involves the use of reduced pressure to increase the flow rate of the mobile phase through a short bed of stationary phase: most of the stationary phase could be used (silica gel, reversed phase material or aluminium oxide) and the technique is applicable to large scale separations. The advantage of this procedure includes its simplicity of equipment, low cost of operation and low solvent consumption, as well as the speed of separation. The disadvantage is that the resolution is only moderate.
5.4 Purification
The purification is the last step and consists in a high-‐resolution separation giving a single pure compound. This procedure involves
30 and High Performance Liquid Chromatography.
5.5 Open Column Chromatography
The gravity-‐driven open column chromatography method is still widely used in natural product chemistry, as it reprsents a rapid and efficent techiques to obtain pure compouds. The separation is based on differential partitioning between the mobile and stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus changing the separation. The main advantage of column chromatography is the relatively low cost and disposability of the stationary phase used in the process. The most used stationary phase is silica gel. The chemical nature of the surface of silica gel consists of exposed silanol groups. These hydroxyl groups are the active centers and potentially can form strong hydrogen bonds with compounds being chromatographed. Thus, in general, the stronger the hydrogen-‐bonding potential of a compound, the stronger it will be retained by silica gel, so that polar compounds are strongly adsorbed, while non-‐polar molecules are poorly or non-‐retained on silica gel. Other stationary phases are aluminium oxide, reversed phase (RP) and Sephadex.
